Point-of-care tenofovir (POC TFV) testing could provide a means of antiretroviral therapy (ART) adherence monitoring for people living with HIV (PLWH), allowing for earlier detection of adherence issues and timely interventions. In this qualitative study, we assessed the acceptability of a POC TFV testing intervention among PLWH and healthcare providers. We conducted in-depth interviews with 20 PLWH and eight healthcare providers participating in the STREAM HIV trial in KwaZulu-Natal, South Africa. We conducted thematic analyses of the acceptability, appropriateness, feasibility, and willingness to use the test, as well as participants’ preferred form of adherence monitoring and differentiated implementation strategies. Participants found POC TFV testing highly acceptable, appropriate, preferable to self-reporting adherence, and feasible to integrate into care. POC TFV testing was well-liked and perceived to have several positive effects. Participants’ desire to impress and build trust with their provider motivated them to take their ART daily to achieve a positive adherence test result at each clinic visit. Nearly all PLWH preferred POC TFV testing over self-reporting their adherence, and all healthcare providers were willing to use the test in routine care. Overall, POC TFV testing may be an acceptable and beneficial tool for improving HIV care and strengthening client-provider relations.